1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding polypeptides that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding polypeptides that confer upon the plant increased growth and/or increased drought, cold, and/or salt tolerance.
2. Background Art
Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity. Crop losses and crop yield losses of major crops such as soybean, rice, maize (corn), cotton, and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on development, growth, and yield of most crop plants are profound. Continuous exposure to drought conditions causes major alterations in the plant metabolism that ultimately lead to cell death and consequently yield losses.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold, and salt tolerance in model drought- and/or salt-tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerant plants using biotechnological methods.
Therefore, what is needed is the identification of the genes and proteins involved in these multi-component processes leading to increased growth and/or increased stress tolerance. Elucidating the function of genes expressed in stress tolerant plants will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement.
There are at least four different signal-transduction pathways leading to stress tolerance in the model plant Arabidopsis thaliana. These pathways are under the control of distinct transcription factors, protein kinases, protein phosphatases and other signal-transduction pathway components (Shinozaki et al., 2000, Curr. Op. Pl. Biol. 3:217-23). These proteins are prime targets for engineering stress tolerance since they could function as master switches; alterations in a single gene would lead to activation of an entire signal-transduction chain leading to stress tolerance.
Sensing of osmotic stress in bacteria as well as in plants is performed by a two-component system comprising a sensing protein and an effecting protein (Wurgler-Murphy S M and Saito S., 1997, Trends in Biochem. Sci. 22:172-6; Shinozaki et al., 2000, Curr. Op. Pl. Biol. 3: 217-23). Mitogen-activated protein kinase-dependent signal transduction pathways are tightly involved in these processes. Another major component of these signal-transduction chains are GTP-binding proteins (G-proteins). Generally speaking, there are at least three classes of G-proteins: a) heterotrimeric (alpha, beta and gamma subunits), b) monomeric (small) proteins, and c) Dyanins. GTP-binding proteins are named as such because each must bind GTP in order to be active. The functions of GTP-binding proteins are varied as they range from directly transmitting an external signal (where the GTP binding protein is associated with a membrane-bound receptor), to participating in vesicle traffic, to importing proteins into sub-cellular compartments.
Monomeric/small G-proteins are involved in many different cellular processes and have been implicated in vesicle traffic/transport systems, cell cycle regulation, and protein import into organelles. To date, more than 200 small G-proteins have been discovered. These proteins may be classified into five superfamilies based on the structural and functional similarities: Ras, Rho/Rac/Cda42, Rab, Sar1/Arf, and Ran. Generally, members of only the Sar1 and Rab families of small G proteins, are involved in vesicle trafficking in yeast (S. cerevisiae) and mammalian cells (Takai et al., 2001, Phys. Rev. 81:153-208). In plants, Rab G proteins have been proven to function in a manner similar to their yeast and mammalian counterparts. Rab G proteins regulate endocytic trafficking pathways and biosynthetic trafficking pathways. Members of the Sar1/Arf family of G proteins also can help recruit coat proteins to transport vesicles (Vemoud et al., 2003, Plant. Physiol. 131:1191-1208).
The SNARE proteins are members of the Rab family of G proteins. The SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor, or SNAP receptor) proteins are cytoplasmically oriented membrane proteins that play a central role in vesicle trafficking and are conserved among yeast and mammals. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs), which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle.
During adaptation to stress, the plant has to recycle its own components, for example, by transporting proteins from one membrane compartment to another, depositing unused proteins in vacuoles, and processing newly synthesized proteins from the ER to the Golgi. Vesicle trafficking has been shown to be actively involved in this biological recycling process under unfavorable environmental conditions. For example, Snsyr1, one of the ABA-related SNARE proteins, plays a role in the stomata movement and root growth (Geelen, D. et al. 2002, Plant Cell 14: 387-406).
Several groups have identified small G-proteins, homologous to the Rab family of small G-proteins, as being induced upon desiccation treatments in plants (Bolte et al., 2000, Plant Mol. Biol. 42:923-36; O'Mahony and Oliver, 1999, Plant Mol. Biol. 39:809-21). These researchers speculate that the small G-proteins could be involved in preservation of membrane integrity or re-structuring upon relief of stress. However, they have not produced transgenic plants with increased stress tolerance by overexpression of these small G-proteins. In addition, Arabidopsis AtRab7, another member of the Rab family of small G proteins, has been shown to be induced after infection of the plant by necrogenic pathogens, and overexpression of AtRab7 has been shown to enhance resistance of transgenic plant to salt and osmotic stress (Mazel et al., 2004 Plant Physiol. 134:118-128).
Although some genes that are involved in stress responses and water use efficiency in plants have been characterized, the characterization and cloning of plant genes that confer stress tolerance and water use efficiency remains largely incomplete and fragmented. For example, certain studies have indicated that drought and salt stress in some plants may be due to additive gene effects, in contrast to other research that indicates specific genes are transcriptionally activated in vegetative tissue of plants under osmotic stress conditions. Although it is generally assumed that stress-induced proteins have a role in tolerance, direct evidence is still lacking, and the functions of many stress-responsive genes are unknown.
There is a fundamental physiochemically-constrained trade-off, in all terrestrial photosynthetic organisms, between CO2 absorption and water loss (Taiz and Zeiger 1991 Plant Physiology, Benjamin/Cummings Publishing Co, p 94). CO2 needs to be in aqueous solution for the action of CO2 fixation enzymes such as Rubisco (Ribulose 1,5-bisphosphate Carboxylase/Oxygenase) and PEPC (Phosphoenolpyruvate carboxylase). As a wet cell surface is required for CO2 diffusion, evaporation will inevitably occur when the humidity is below 100% (Taiz and Zeiger 1991 Plant Physiology, Benjamin/Cummings Publishing Co p 257). Plants have numerous physiological mechanisms to reduce water loss (e.g. waxy cuticles, stomatal closure, leaf hairs, sunken stomatal pits). As these barriers do not discriminate between water and CO2 flux, these water conservation measures will also act to increase resistance to CO2 uptake (Kramer 1983 Water Relations of Plants, Academic Press p 305). Photosynthetic CO2 uptake is absolutely required for plant growth and biomass accumulation in photoautotrophic plants. Water Use Efficiency (WUE) is a parameter frequently used to estimate the trade off between water consumption and CO2 uptake/growth (Kramer 1983 Water Relations of Plants, Academic Press p 405). WUE has been defined and measured in multiple ways. One approach is to calculate the ratio of whole plant dry weight, to the weight of water consumed by the plant throughout its life (Chu et al., 1992, Oncologia, 89:580). Another variation is to use a shorter time interval when biomass accumulation and water use are measured (Mian et al., 1998, Crop Sci. 38:390). Often measurements from restricted parts of the plant are used, for example, measuring only aerial growth and water use (Nienhuis et al., 1994, Amer. J. Bot. 81:943). WUE has also been defined as the ratio of CO2 uptake to water vapor loss from a leaf or portion of a leaf, often measured over a very short time period (seconds/minutes) (Kramer 1983, Water Relations of Plants, Academic Press p. 406). The ratio of 13C/12C fixed in plant tissue, and measured with an isotope ratio mass-spectrometer, has also been used to estimate WUE in plants using C3 photosynthesis (Martin et al., 1999, Crop Sci. 1775).
An increase in WUE is informative about the relatively improved efficiency of growth and water consumption, but on it's own it doesn't describe which of these two processes (or both) have changed. In selecting traits for improving crops, an increase in WUE due to a decrease in water use, without a change in growth would have particular merit in an irrigated agricultural system where the water input costs were high. An increase in WUE driven mainly by an increase in growth without a corresponding jump in water use would have applicability to all agricultural systems. In many agricultural systems where water supply is not limiting, an increase in growth, even if it came at the expense of an increased water use (i.e. no change in WUE), could also increase yield. Therefore new methods to increase both WUE and biomass accumulation are required to improve agricultural productivity. As WUE integrates many physiological processes relating to primary metabolism and water use, it is typically a highly polygenic trait with a large genotype by environment interaction (Richards et al., 2002, Crop Sci. 42:111). For these and other reasons few attempts to select for WUE changes in traditional breeding programs have been successful.
There is a need, therefore, to identify genes expressed in stress tolerant plants and plants that are efficient in water use that have the capacity to confer stress tolerance and water use efficiency to its host plant and to other plant species. Newly generated stress tolerant plants will have many advantages, such as an increased range in which the crop plants can be cultivated, by for example, decreasing the water requirements of a plant species. Other desirable advantages include increased resistance to lodging, the bending of shoots or stems in response to wind, rain, pests, or disease.